Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Phage-mediated counting by the naked eye of miRNA molecules at attomolar concentrations in a Petri dish

Nature Materials volume 14, pages 1058–1064 (2015)Cite this article

Subjects

Abstract

The ability to count biomolecules such as cancer-biomarker miRNAs with the naked eye is seemingly impossible in molecular diagnostics. Here, we show an ultrasensitive naked-eye-counting strategy for quantifying miRNAs by employing T7 phage—a bacteria-specific virus nanoparticle—as a surrogate. The phage is genetically engineered to become fluorescent and capable of binding a miRNA-capturing gold nanoparticle (GNP) in a one-to-one manner. Target miRNAs crosslink the resultant phage–GNP couple and miRNA-capturing magnetic microparticles, forming a sandwich complex containing equimolar phage and miRNA. The phage is then released from the complex and developed into one macroscopic fluorescent plaque in a Petri dish by plating it in a host bacterial medium. Counting the plaques by the naked eye enables the quantification of miRNAs with detection limits of ∼3 and ∼5 aM for single-target and two-target miRNAs, respectively. This approach offers ultrasensitive and convenient quantification of disease biomarkers by the naked eye.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: General concepts of the counting strategy.
Figure 2: Preparation and characterization of T7–GNP probes.
Figure 3: Determination of both single-target and multiple-target miRNAs by the counting strategy.
Figure 4: Quantification of various real samples (cells, serum and tissues) by the counting strategy.

Similar content being viewed by others

References

  1. Hamburg, M. A. & Collins, F. S. The path to personalized medicine. New Engl. J. Med. 363, 301–304 (2010).

    Article  CAS  Google Scholar 

  2. Taton, T. A., Mirkin, C. A. & Letsinger, R. L. Scanometric DNA array detection with nanoparticle probes. Science 289, 1757–1760 (2000).

    Article  CAS  Google Scholar 

  3. Valentini, P. et al. Gold-nanoparticle-based colorimetric discrimination of cancer-related point mutations with picomolar sensitivity. ACS Nano 7, 5530–5538 (2013).

    Article  CAS  Google Scholar 

  4. Cao, Y. C., Jin, R. & Mirkin, C. A. Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection. Science 297, 1536–1540 (2002).

    Article  CAS  Google Scholar 

  5. Qiu, F., Jiang, D., Ding, Y., Zhu, J. & Huang, L. L. Monolayer-barcoded nanoparticles for on-chip DNA hybridization assay. Angew. Chem. Int. Ed. 47, 5009–5012 (2008).

    Article  CAS  Google Scholar 

  6. Zhou, X., Cao, P., Tian, Y. & Zhu, J. Expressed peptide assay for DNA detection. J. Am. Chem. Soc. 132, 4161–4168 (2010).

    Article  CAS  Google Scholar 

  7. Nam, J.-M., Stoeva, S. I. & Mirkin, C. A. Bio-bar-code-based DNA detection with PCR-like sensitivity. J. Am. Chem. Soc. 126, 5932–5933 (2004).

    Article  CAS  Google Scholar 

  8. Stoeva, S. I., Lee, J.-S., Smith, J. E., Rosen, S. T. & Mirkin, C. A. Multiplexed detection of protein cancer markers with biobarcoded nanoparticle probes. J. Am. Chem. Soc. 128, 8378–8379 (2006).

    Article  CAS  Google Scholar 

  9. Mao, C. et al. Viral assembly of oriented quantum dot nanowires. Proc. Natl Acad. Sci. USA 100, 6946–6951 (2003).

    Article  CAS  Google Scholar 

  10. Mao, C. et al. Virus-based toolkit for the directed synthesis of magnetic and semiconducting nanowires. Science 303, 213–217 (2004).

    Article  CAS  Google Scholar 

  11. Liu, A., Abbineni, G. & Mao, C. Nanocomposite films assembled from genetically engineered filamentous viruses and gold nanoparticles: Nanoarchitecture- and humidity-tunable surface plasmon resonance spectra. Adv. Mater. 21, 1001–1005 (2009).

    Article  CAS  Google Scholar 

  12. Mao, C., Wang, F. & Cao, B. Controlling nanostructures of mesoporous silica fibers by supramolecular assembly of genetically modifiable bacteriophages. Angew. Chem. Int. Ed. 51, 6411–6415 (2012).

    Article  CAS  Google Scholar 

  13. Wang, F., Nimmo, S., Cao, B. & Mao, C. B. Oxide formation on biological nanostructures via a structure-directing agent: Towards an understanding of precise transcription. Chem. Sci. 3, 2639–2645 (2012).

    Article  CAS  Google Scholar 

  14. Eber, F. J., Eiben, S., Jeske, H. & Wege, C. Bottom-up-assembled nanostar colloids of gold cores and tubes derived from tobacco mosaic virus. Angew. Chem. Int. Ed. 52, 7203–7207 (2013).

    Article  CAS  Google Scholar 

  15. Cao, B., Zhu, Y., Wang, L. & Mao, C. B. Controlled alignment of filamentous supramolecular assemblies of biomolecules into centimeter-scale highly ordered patterns by using nature-inspired magnetic guidance. Angew. Chem. Int. Ed. 52, 11750–11754 (2013).

    Article  CAS  Google Scholar 

  16. Mao, C., Liu, A. & Cao, B. Virus-based chemical and biological sensing. Angew. Chem. Int. Ed. 48, 6790–6810 (2009).

    Article  CAS  Google Scholar 

  17. Mohan, K., Donavan, K. C., Arter, J. A., Penner, R. M. & Weiss, G. A. Sub-nanomolar detection of prostate-specific membrane antigen in synthetic urine by synergistic, dual-ligand phage. J. Am. Chem. Soc. 135, 7761–7767 (2013).

    Article  CAS  Google Scholar 

  18. Cuervo, A. et al. Structural characterization of the bacteriophage T7 tail machinery. J. Biol. Chem. 288, 26290–26299 (2013).

    Article  CAS  Google Scholar 

  19. Chan, L. Y., Kosuri, S. & Endy, D. Refactoring bacteriophage T7. Mol. Syst. Biol. 1, 2005.0018 (2005).

    Article  Google Scholar 

  20. Slootweg, E. J. et al. Fluorescent T7 display phages obtained by translational frameshift. Nucleic Acids Res. 34, e137 (2006).

    Article  Google Scholar 

  21. Tsuboyama, M. & Maeda, I. Combinatorial parallel display of polypeptides using bacteriophage T7 for development of fluorescent nano-bioprobes. J. Biosci. Bioeng. 116, 28–33 (2013).

    Article  CAS  Google Scholar 

  22. Huang, Y. et al. Programmable assembly of nanoarchitectures using genetically engineered viruses. Nano Lett. 5, 1429–1434 (2005).

    Article  CAS  Google Scholar 

  23. Garcia-Doval, C. & Raaij, M. J. V. Structure of the receptor-binding carboxy-terminal domain of bacteriophage T7 tail fibers. Proc. Natl Acad. Sci. USA 109, 9390–9395 (2012).

    Article  CAS  Google Scholar 

  24. Dunn, J. J. & Studier, F. W. Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J. Mol. Biol. 166, 477–535 (1983).

    Article  CAS  Google Scholar 

  25. Johnson, S. M. et al. RAS is regulated by the let-7 microRNA family. Cell 120, 635–647 (2005).

    Article  CAS  Google Scholar 

  26. Esquela-Kerscher, A. & Slack, F. J. Oncomirs—microRNAs with a role in cancer. Nature Rev. Cancer 6, 259–269 (2006).

    Article  CAS  Google Scholar 

  27. Lin, P. Y., Yu, S. L. & Yang, P. C. MicroRNA in lung cancer. Br. J Cancer 103, 1144–1148 (2010).

    Article  CAS  Google Scholar 

  28. Bousquet, M., Harris, M. H., Zhou, B. & Lodish, H. F. MicroRNA miR-125b causes leukemia. Proc. Natl Acad. Sci. USA 107, 21558–21563 (2010).

    Article  CAS  Google Scholar 

  29. Monya, B. qPCR: Quicker and easier but don’t be sloppy. Nature Methods 8, 207–212 (2011).

    Article  Google Scholar 

  30. Kumar, M. S. et al. Suppression of non-small cell lung tumor development by the let-7 microRNA family. Proc. Natl Acad. Sci. USA 105, 3903–3908 (2008).

    Article  CAS  Google Scholar 

  31. Serwer, P. & Pichler, M. E. Electrophoresis of bacteriophage T7 and T7 capsids in agarose gels. J. Virol. 28, 917–928 (1978).

    CAS  Google Scholar 

  32. Xu, T. et al. MicroRNA-195 suppresses tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells. Hepatology 50, 113–121 (2009).

    Article  CAS  Google Scholar 

  33. Jusufović, E. et al. let-7b and miR-126 are down-regulated in tumor tissue and correlate with microvessel density and survival outcomes in non-small-cell lung cancer. PLoS ONE 7, e45577 (2012).

    Article  Google Scholar 

  34. Liu, Z. L., Wang, H., Liu, J. & Wang, Z. X. MicroRNA-21 (miR-21) expression promotes growth, metastasis, and chemo- or radioresistance in non-small cell lung cancer cells by targeting PTEN. Mol. Cell Biochem. 372, 35–45 (2013).

    Article  CAS  Google Scholar 

  35. Thellin, O. et al. Housekeeping genes as internal standards: Use and limits. J. Biotechnol. 75, 291–295 (1999).

    Article  CAS  Google Scholar 

  36. Vandesompele, J. et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, research0034 (2002).

    Article  Google Scholar 

  37. Dheda, K. et al. Validation of housekeeping genes for normalizing RNA expression in real-time PCR. BioTechniques 37, 112–119 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Key Basic Research Program of China (2011CB933503), the Special Funds of the National Natural Science Foundation of China for Basic Research Projects of Scientific Instruments (61127002), the Basic Research Program of Jiangsu Province (BK2011036) and the Jiangsu Province Funds for Distinguished Young Scientists (BK20140049). Y.Z. and C.M. are grateful for financial support from the National Institutes of Health (EB015190 and CA200504), the National Science Foundation (CMMI-1234957), the Department of Defense Congressionally Directed Medical Research Programs, the Oklahoma Center for Adult Stem Cell Research (434003) and the Oklahoma Center for the Advancement of Science and Technology (HR14-160).

Author information

Author notes

  1. Xin Zhou and Peng Cao: These authors contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Bioelectronics, Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China

    Xin Zhou & Ning Gu

  2. Laboratory of Cellular and Molecular Biology, Jiangsu Branch of China Academy of Chinese Medical Sciences, Nanjing 210028, China

    Peng Cao & Wuguang Lu

  3. Department of Chemistry and Biochemistry, Stephenson Life Sciences Research Center, University of Oklahoma, 101 Stephenson Parkway, Norman, Oklahoma 73019-5300, USA,

    Ye Zhu & Chuanbin Mao

  4. School of Materials Science and Engineering, Zhejiang University, Hangzhou, Zhejiang 310027, China

    Chuanbin Mao

Authors
  1. Xin Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peng Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ye Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wuguang Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ning Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Chuanbin Mao
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

X.Z. and C.M conceived the experiments. X.Z., P.C., C.M. and Y.Z. performed the experiments. W.L. assisted with the experiments, X.Z., Y.Z. and C.M. wrote the manuscript and analysed the data. N.G. and C.M. designed and supervised the project. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Ning Gu or Chuanbin Mao.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 4632 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhou, X., Cao, P., Zhu, Y. et al. Phage-mediated counting by the naked eye of miRNA molecules at attomolar concentrations in a Petri dish. Nature Mater 14, 1058–1064 (2015). https://doi.org/10.1038/nmat4377

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmat4377

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing